The present invention relates to a chemically synthesised and theoretically designed promoter for high level expression of transgenes in different organisms and a method for designing of the said promoter. The invention further relates to testing of the said promoter to demonstrate high level activity as compared to the natural Cauliflower Mosaic Virus (CaMV) 35S promoter.
The invention emphasises the development of an artificial DNA sequence on the basis of computational analysis of various genes which express at high level in plants. The invention provides a new outlook in the field of developing artificial transcriptional regulatory elements that act in cis on genes. The present invention takes pride in claiming that DNA elements that function as efficient regulatory sequences in the cells of higher organisms can be designed and synthesised to achieve desired level of expression on the basis of knowledge deduced from computational biology and bioinformatics.
One of the main objectives of plant genetic engineering is to develop transgenic plants with new characteristics and traits, which may include insect resistance, virus resistance, herbicide resistance, yield enhancement, stress tolerance, nutritional improvement, expression of industrially valuable proteins in economically profitable expression systems, like plants, etc. Many factors contribute to high level expression of genes which code for such desired characteristics, where xe2x80x98expressionxe2x80x99 includes transcription, translation and post translational events. The abundance of any one transcript in a cell directly relates to transcriptional events, which in turn depends upon the strength of the promoter from which it is expressed. Thus, for the development of transgenic plants where high level of transgene expression is to be obtained, it becomes absolutely indispensable that the transgene be expressed from a strong promoter, the transcript is stable, it is translated efficiently and that the resultant protein is also stable in plant cell. Each of these steps synergistically contributes to enhancing the level of expression of the product of the transgene.
A promoter can be defined as a pool of cis-acting elements, which work in co-ordination with trans acting transcriptional factors to achieve expression of the gene attached to it. A promoter provides an efficient docking site for RNA polymerase and the related accessory proteins, which in turn contribute to the transcription of the gene situated operably therewith. Thus, as mentioned, promoters are highly specialised DNA sequences which govern the time and efficiency of transcription. A promoter is classified as a constitutive promoter when it is operable almost equally at all times in a given organism, for example, the CaMV 35S promoter. Other promoters are tissue specific or inducible. The strength of a promoter varies depending on the frequency of initiation of transcriptional events. Depending on strength, promoters can further be classified as strong or weak.
Different types of promoters are required in plant biotechnology, depending upon the target use. Constitutive high level expression promoters are most useful to develop transgenic plants for high level production of commercially required proteins. Such high level expression is also desirable in several situations for modifying metabolic pathways and for improving plants to withstand a variety of stress situations.
Previous reports mainly deal with the identification of natural promoter elements in genes and their improvement. These include, the identification of the CaMV 35S promoter by Odell et al., Nature 313: 810-812 (1985), who had shown the strength and constitutive nature of CaMV 35S promoter. Later, Jensen et al., Nature 321: 669-674 (1986), Jefferson et ai., EMBOJ., 6: 390-3907 (1987), and Sander et al., Nucleic Acids Research, 4: 1543-1558 (1987), showed measurable levels of reporter gene mRNA expressed from 35S CaMV promoter in extracts prepared from leaves, stems, roots and flowers of transgenic plants. The CAMV 35S promoter has been widely used by scientists in the field of plant genetic engineering. Morelli et al., Nature (1985) 315:200-204 described that the CaMV 35S promoter is transcribed at a relatively high rate as evidenced by a ten-fold increase in transcription products as compared to the NOS promoter. Abel et al., Science (1986) 232:738-743, Bevan et al., EMBO J. (1985) 4:1921-1926, Morelli et al., Nature (1985) 315:200-204, and Shah et al., Science (1986) 233:478-481 described that the 35S CaMV promoter is moderately strong and constitutively active. Therefore, the CaMV 35S promoter has been used to express a number of foreign genes in transgenic plants. Odell et al., Nature 313: 810-812 (1985), described that initiation of transcription from the 35S promoter is dependent on proximal sequences, which included a TATA element, while the rate of transcription was determined by sequences that were dispersed over 300 bp of upstream DNA. Simpson et al., Nature 323:551-554 (1986) described this region as an enhancer region (sequences which activate transcription are termed enhancers).
Subsequently, other workers tried to improve the CaMV promoter. Kay et al. Science 236: 1299-1302 (1987) duplicated a large region (253 bp) of the naturally existing CaMV 35S promoter and reported enhancement in its activity. Odell, et al., Plant Mol. Biol. 10:263-272 (1988), reported the use of a part of the CaMV 35S promoter as an enhancer in the nopaline synthase promoter. Mitsuhara, et al., Plant Cell Physiol. 37 (1): 49-59 (1996) compared many combinations of different CaMV 35S promoter sequence elements. By increasing the number of repeats of the native enhancer element, they obtained enhanced expression of the reporter gene. Ni, et al., The Plant Journal 7(4):661-676 (1995) combined portions of the naturally occurring octopine and mannopine synthase promoters to develop an efficient chimeric promoter. Ellis, et al., EMBO 6:11-16 (1987), reported the use of a natural octopine synthase promoter fragment to enhance the activity of the maize (adh-1) gene.
Other developments include identification of other natural promoter elements for expression of genes in plants. These include the use of the Figwort Mosaic Virus promoter for achieving enhanced expression per U.S. Pat. No. 5,378,619, Rubisco promoter as per U.S. Pat. No. 4,962,028, chimeric CaMV enhanced mannopine synthase promoter as per U.S. Pat. No. 5,106,739, enhanced CaMV 35S promoter as per U.S. Pat. No. 5,322,938, and the glutamine synthetase promoter for organ specific expression in plants as per U.S. Pat. No. 5,391,725.
As of now, attempts have been made to identify the naturally existing promoter sequences to be used as such or to exchange or rearrange parts of natural promoters so as to achieve a higher level of expression. However, in no case an attempt has been made to design an artificial promoter based on knowledge gained from computational analysis of various DNA sequences present upstream of the gene sequence, reported in the database.
Some of the objectives of this invention are to design a synthetic promoter aimed at achieving the desired level of expression of the target genes in plant cells, but also in bacteria, yeast, lower euckaryotic cells and animal cells. to use such a promoter in combination with specific regulatory elements, to modify it appropriately so as to make it tissue specific, development stage specific, organ specific and or inducible by specific external environmental/applied factors, as well as, providing, a new approach for studying the complexity of the interaction between cis-acting elements and trans-acting factors.
The present invention relates to analysing the gene sequence database for designing promoters for achieving the desired level of expression of transgenes in different organisms and a method for synthesis of the designed promoter. The invention further relates to testing and demonstrating high level of activity of the synthetic promoter as compared to the natural CaMV 35S promoter.
As an example, the invention demonstrates the designing of an artificial DNA sequence on the basis of computational analysis of various genes which express at high level in plants. The invention provides a new look in the field of synthesizing designer/custom made transcriptional regulatory elements. The approach includes the identification of DNA sequences representing, minimal promoter (SEQ ID NO 2, 3 and 5), conserved domain I and its sub domains a, b and c (SEQ ID NO 6), transcription start site context (SEQ ID NO.4), conserved domain II and its sub domains a, b, c and d (SEQ ID NO 7,8,9 and 10), conserved domain III (SEQ ID NO 11), domain between TATA and TS(SEQ ID NO 12), 5xe2x80x2untranslated leader (SEQ ID NO 13), translational initiation codon contexts (SEQ ID NO 14 and 15) that act cis on the gene and N-terminal amino acids (SEQ ID NO 16) that may give stability to proteins. An example of such a construct designed in this study is SEQ ID NO: 1. The present invention takes pride in claiming that DNA elements that function as efficient promoter regulatory sequences in a variety of tissues and in a wide spectrum of organisms can be designed on the basis of knowledge generated from computational biology and bioinformatics and synthesised. This invention shows that a biological active and efficiently functional promoter can be synthesised to express in even the most complex organisms.